1. Field of the Invention
The present invention relates to sulfohydrolases, such as galactan sulfohydrolases, such as nu- and mu-carrageenan sulfohydrolases. The present invention is directed to the amino acid and nucleotide sequences of sulfohydrolases. The present invention is further directed to enzymatic modification of sulfated compounds, such as galactans. For example, the enzymatic modification may involve tailoring of the properties of sulfated galactans, such as gelling properties, such as by removal of sulfate groups and creation of a bridge between ring positions in a saccharide structure of the galactan. The present invention is further directed to processes of extracting nu-carrageenan from seaweed. The present invention is also directed to enzymatically modified compounds.
2. Discussion of Background
Hydrocolloids, which may be broadly defined as substances that yield a gel in the presence of water, are used in part for their rheological properties, and may also provide benefits in stability.
There are several classes of hydrocolloids. One categorization approach breaks these classes into exudates, such as gum arabic, ghatti, karaya, talha, and tragacanth; extracts, such as alginate from brown seaweeds, agar, carrageenan, and furcelleran from red seaweeds, and konjak (glucomannan), guar, pectin and arabinogalactan from land plants; biopolymers, such as xanthan; chemically modified hydrocolloids, such as the cellulosics, including carboxymethyl cellulose, hydroxypropyl cellulose, and carboxymethylhydroxymethyl cellulose; and intermediate forms, such as microcrystalline cellulose.
Red seaweeds are known sources of industrial gelling and thickening cell-wall sulfated galactans referred to as agar and carrageenans. They consist of a linear backbone of galactopyranose residues linked by alternating alpha(1xe2x86x923) and beta(1xe2x86x924) linkages. While all xcex2-linked residues are in the D-configuration, the alpha(1xe2x86x924)-linked galactose units are in the L-configuration in agars and in the D-configuration in carrageenans.
Agar is extracted from dried algae by more or less hot alkaline solutions (100-120xc2x0 C.). After filtration, agar solutions are allowed to gel de-watered by pressing, dried, and ground, as disclosed in ARMISEN et al., xe2x80x9cProduction, Properties and Uses of Agarxe2x80x9d, Production and Utilization of Products from Commercial Seaweeds, FAO Fisheries Technical Paper, 288, pp. 1-57 (1987), the disclosure of which is herein incorporated by reference in its entirety. Seaweed sources for agar extraction include the genera Gelidium, Pterocladia, Gelidiella, and Gracilaria, as disclosed in STANLEY, xe2x80x9cProduction, Properties and Uses of Carrageenanxe2x80x9d, Production and Utilization of Products from Commercial Seaweeds, FAO Fisheries Technical Paper, 288, pp. 116-146 (1987), the disclosure of which is herein incorporated by reference in its entirety.
Carrageenan is itself a generic name for a family of natural water-soluble sulfated galactans isolated from red seaweeds. The thickening and gelation properties exhibited by carrageenans are useful in food and cosmetic formulations, as disclosed in THERKELSEN, xe2x80x9cCarrageenanxe2x80x9d, Industrial Gums: Polysaccharides and their Derivatives, 3rd ed., pp. 145-180, (1993), and DeRUITER et al., xe2x80x9cCarrageenan Biotechnologyxe2x80x9d, Trends in Food Science and Technology, Vol. 8, pp. 389-395 (1997), both of which are herein incorporated by reference in their entireties.
Seaweed sources for carrageenan include the genera Eucheuma (such as E. spinosum, E. cottonii (=Kappaphycus alvarezii), and E. denticulatum), Chondrus (such as C. crispus), Calliblepharis (such as C. jubata), and Gigartina (such as G. radula and G. stellata).
Carrageenans are linear, partially sulfated galactans mainly composed of repeating dimers of an alpha(1-4)-linked D-galactopyranose or 3,6-anhydro-D-galactopyranose residue and a beta(1-3)-linked D-galactopyranose residue. As noted above, agars are likewise linear, partially sulfated galactans of similar structure, except that the alpha(1-4)-linked galactopyranose residue is in the L-form. Carrageenan occurs in several structures that differ primarily in the number and placement of sulfate groups on the dimer backbone, and in whether the individual residues of the dimer are present in the left hand (4C1) or right-hand (1C4) xe2x80x98chairxe2x80x99 configuration. These structures include kappa (xcexa), iota ("igr"), lambda (xcex), theta (xcex8), mu (xcexc), and nu (xcexd), as shown below. The iota-, kappa-, and theta-carrageenans contain 3,6-anhydro bridges, whereas the nu-, mu-, and lambda-carrageenans do not have this bridge. Furthermore, the conformation of the alpha-linked unit of nu-, mu-, and lambda-carrageenans is different from the anhydrobridge-containing carrageenans, preventing sufficient helix aggregation. Helix aggregation is important because helices facilitate formation of gels. In this regard, kappa-carrageenan forms firm, brittle gels, whereas iota-carrageenan forms elastic, soft gels, and whereas lambda-carrageenan is a non-gelling thickening agent. 
The amount of SO3xe2x88x92 in carrageenans can be considerable and vary between 0 and 41% (w/w), resulting in highly negatively charged polymers. Ideal kappa-, iota-, and lambda-carrageenan dimers respectively have 1, 2, and 3 sulfate esters groups, resulting in typical sulfate contents of respectively 22%, 32%, and 38% (w/w). However, large variations in sulfate can occur in commercial extracts due to differences in seaweed species or batches. The sulfate ester linkages are chemically very stable, and there are no apparent or practical chemical methods to modify the sulfate level or distribution without also lowering the molecular weight of the polymer, except for the removal of 6-O-sulfate from precursor carrageenans as is done during alkaline treatment.
Carrageenan is typically extracted commercially from red seaweeds by boiling in aqueous solution, sometimes under alkaline conditions, followed by filtration, concentration, precipitation, and drying. Precipitation may either be by alcohol addition, or by gelling with salts followed by pressing of the gel, as discussed in STANLEY, xe2x80x9cProduction, Properties and Uses of Carrageenanxe2x80x9d, Production and Utilization of Products from Commercial Seaweeds, FAO Fisheries Technical Paper, 288, pp. 116-146 (1987), the disclosure of which is herein incorporated by reference in its entirety. Semi-refined carrageenans are also produced by treating seaweeds with alkali followed by thorough rinsing with water. These treatments improve the gelling characteristics of the carrageenan preparation and remove most of the proteins, pigments and small metabolites; such preparations also contain other polymers such as cellulosic materials, as discussed in HOFFMANN et al., xe2x80x9cEffect of Isolation Procedures on the Molecular Composition and Physical Properties of Eucheuma Cottonii Carrageenanxe2x80x9d, Food Hydrocolloids, 9, pp. 281-289 (1995), the disclosure of which is herein incorporated by reference in its entirety.
The non-gelling mu- and nu-carrageenans are the natural precursors present in seaweed of, respectively, kappa- and iota-carrageenans, and have a sulfate ester group at the C-6 position of the alpha(1-4)-linked D-galactopyranose residue of the dimeric unit. It has been generally assumed that elimination of the sulfate from the C-6 sulfate ester of the precursors, and formation of the 3,6-anhydro bridge, occur concomitantly during the strong alkaline treatment.
The functional properties (including helix formation, rheological properties, and applications) of the different carrageenans are determined by (1) molecular weight of the polymer (2) the number of sulfate ester groups and their place of substitution of the carbon backbone, and (3) the number of 3,6-anhydro-galactose residues, as discussed in THERKELSEN, xe2x80x9cCarrageenanxe2x80x9d, Industrial Gums: Polysaccharides and their Derivatives, 3rd edition, pp. 145-180 (1993); VIEBKE et al., xe2x80x9cCharacterization of Kappa- and Iota-Carrageenan Coils and Helices by MALLS/GPCxe2x80x9d, Carbohydr. Polym., Vol. 27, pp. 145-154 (1995); and Le QUESTEL et al., xe2x80x9cComputer Modelling of Sulfated Carbohydrates: Applications to Carrageenansxe2x80x9d, Int. J. Biol. Macromol., Vol. 17, pp. 161-174 (1995), the disclosures of which are herein incorporated by reference in their entireties.
Alkali treatments allow some control over the ratio of carrageenan forms in the final product. However, it is often difficult to predict and obtain the desired final product.
It has been reported that the red seaweed Porphyra umbilicalis, contains a xe2x80x9csulfohydrolasexe2x80x9d which catalyzes the release of sulfate from porphyran, the major polysaccharide from Porphyra spp. related to agar in that it contains about 10% (w/w) of 3,6-anhydro-L-galactose. It also contains L-galactose-6-sulfate. These latter units can be converted into 3,6-anhydro-L-galactose by the action of an enzyme partially purified from an extract of the parent seaweed as discussed in REES, xe2x80x9cEnzymic Synthesis of 3:6-Anhydro-L-Galactose within Porphyran from L-Galactose 6-Sulphate Unitsxe2x80x9d, Biochem. J., 81, pp. 347-352 (1961); and REES, xe2x80x9cEnzymatic Desulphation of Porphyranxe2x80x9d, Biochem. J., 80, pp. 449-453 (1961), the disclosures of which are both incorporated herein by reference in their entireties.
It has also been reported that carrageenan-producing red seaweeds contain a xe2x80x9csulfohydrolasexe2x80x9d which catalyzes the release of sulfate from carrageenan precursors. The sulfohydrolase in Chondrus crispus is discussed in WONG et al., xe2x80x9cSulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)xe2x80x9d, Plant Physiology, Vol. 61, pp. 663-666 (1978), the disclosure of which is herein incorporated by reference in its entirety. The sulfohydrolase in Calliblepharis jubata is discussed in ZINOUN et al., xe2x80x9cEvidence of Sulfohydrolase Activity in the Red Alga Calliblepharis jubataxe2x80x9d, Botanica Marina, Vol. 40, pp. 49-53 (1997), the disclosure of which is herein incorporated by reference in its entirety. The sulfohydrolase in Gigartina stellata is discussed in both LAWSON et al., xe2x80x9cAn Enzyme for the Metabolic Control of Polysaccharide Conformation and Functionxe2x80x9d, Nature, Vol. 227, pp. 392-93 (July 25, 1970) and WONG et al., xe2x80x9cSulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)xe2x80x9d, Plant Physiology, Vol. 61, pp. 663-666 (1979), the disclosures of which are herein incorporated by reference in their entireties. Such enzymes have not been previously purified to homogeneity and no electrophoresis data has been provided in previous reports.
The mode of action and degree of specificity of galactan sulfohydrolases have only been described in general terms in the literature. As discussed in CRAIGIE et al., xe2x80x9cCarrageenan Biosynthesisxe2x80x9d, Proc. Int. Seaweed Symp., pp. 369-377 (1978), the disclosure of which is herein incorporated by reference, it is unclear how many sulfohydrolases with different specificity are present in C. crispus. In general, there is little discussion in the literature of the chemical structure of the sulfohydrolase substrates, the end-products of enzymatic action, and the extent of action of the sulfohydrolase on the galactan precursor.
Thus, there is a need for a variety of sulfohydrolases with different specificity which can be used to tailor the properties of sulfated compounds, such as galactans.
The present invention is directed to providing purified sulfohydrolases having various specificities.
The present invention is also directed to amino acid and nucleotide sequences of sulfohydrolases.
The present invention is directed to methods of enzymatically modifying sulfated compounds.
The present invention is additionally directed to methods of extracting carrageenan from seaweed.
The present invention is further directed to sulfated compounds, such as galactans, which have been modified with sulfohydrolases, such as to modify gelling properties.
In accordance with one aspect, the present invention is directed to sulfohydrolase having a purity level based on total amount of protein of at least about 40 wt %. The purity level may also be at least about 70 wt %, at least about 90 wt %, or at least about 95 wt %.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence of SEQ ID NO: 17.
In accordance with yet another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence having at least about 25% homology with SEQ ID NO: 17. The homology with SEQ ID NO: 17 may also be at least about 50%, at least about 80%, or at least about 90%.
In accordance with still another aspect, the present invention is directed to an isolated nucleic acid sequence which will hybridize under hybridization conditions with a nucleic acid of SEQ ID NO: 17.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence of SEQ ID NO: 18.
In accordance with yet another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence of SEQ ID NO: 19.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence of SEQ ID NO: 20.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence of SEQ ID NO: 21.
In accordance with yet another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence having at least about 25% homology with SEQ ID NO: 18. The homology with SEQ ID NO: 18 may also be at least about 50%, at least about 80%, or at least about 90%.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence having at least about 90% homology with SEQ ID NO: 19.
In accordance with still another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence having at least about 90% homology with SEQ ID NO: 20.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence comprising a sequence having at least about 90% homology with SEQ ID NO: 21.
In accordance with yet another aspect, the present invention is directed to an isolated nucleic acid sequence which will hybridize under hybridization conditions with a nucleic acid of SEQ ID NO: 18.
In accordance with another aspect, the present invention is directed to an isolated nucleic acid sequence encoding for an amino acid sequence corresponding to SEQ ID NO: 22.
In accordance with yet another aspect, the present invention is directed to an isolated protein comprising an amino acid sequence comprising SEQ ID NO: 22.
In accordance with still another aspect, the present invention is directed to an isolated protein comprising an amino acid sequence which has at least about 35% homology with SEQ ID NO: 22. The homology with SEQ ID NO: 22 may also be at least about 50% or at least about 80%.
In accordance with yet another aspect, the present invention is directed to an isolated nucleic acid sequence encoding for an amino acid sequence corresponding to SEQ ID NO: 23.
In accordance with another aspect, the present invention is directed to an isolated protein comprising an amino acid sequence comprising SEQ ID NO: 23.
In accordance with yet another aspect, the present invention is directed to an isolated protein comprising an amino acid sequence which has at least about 20% homology with SEQ ID NO: 23. The homology with SEQ ID NO: 23 may also be at least about 50% or at least about 80%.
In accordance with a further aspect, the present invention is directed to a process for purifying at least one sulfohydrolase, comprising: subjecting an extract from seaweed to fractionation to obtain fractions; and subjecting at least one of the fractions to phenyl sepharose chromatography to obtain sepharose fractions containing at least one sulfohydrolase.
In accordance with still another aspect, the present invention is directed to an enzymatically modified compound which has been modified by an isolated sulfohydrolase having a purity level based on total amount of protein of at least about 40 wt %. The purity of the isolated sulfohydrolase may be at least about 70 wt %, at least about 90 wt %, or at least about 95 wt %.
In accordance with a further aspect, the present invention is directed to a process of enzymatically modifying a sulfated compound, comprising: combining at least one sulfohydrolase, having a purity level based on total amount of protein of at least about 40 wt %, with a sulfated compound to form a reaction mixture; and incubating the reaction mixture to remove sulfate groups from the sulfated compound to form an enzymatically modified compound.
In accordance with still another aspect, the present invention is directed to a process of enzymatically modifying a sulfated compound, comprising: incubating a first sulfohydrolase with a sulfated compound to remove sulfate groups from the sulfated compound to form an intermediate compound; and subsequently incubating the intermediate compound with a second sulfohydrolase to remove sulfate groups to form an enzymatically modified compound.
In accordance with another aspect, the present invention is directed to a product made by incubating a sulfated compound with a solution having a protein content consisting essentially of a sulfohydrolase which removes sulfate groups processively.
In accordance with still another aspect, the present invention is directed to a product made by incubating a sulfated compound with a solution having a protein content consisting essentially of a sulfohydrolase which removes sulfate groups randomly.
In accordance with yet another aspect, the present invention is directed to a method for extracting one of nu- and mu-carrageenan from seaweed, comprising: dispersing seaweed in a salt solution comprising K2CO3 to form a dispersion; filtering the dispersion to obtain a liquid; ultrafiltering the dispersion to remove salts; concentrating the liquid; adjusting the pH of the liquid to about 8 to 8.5; and precipitating the one of nu- and mu-carrageenan from the liquid.
In one aspect, the sulfohydrolase is capable of removing sulfate from hydrocolloid. The hydrocolloid may be one of glycosaminoglycan, fucan, and galactan. In other words, the sulfohydrolase hydrolyzes ester-sulfate bonds present in hydrocolloids.
In another aspect, the sulfohydrolase is capable of removing sulfate from galactan.
In another aspect, the galactan comprises carrageenan. The carrageenan may comprise mu-carrageenan, e.g., comprising at least about 50 mol % mu-carrageenan. The carrageenan may comprise nu-carrageenan, e.g., comprising at least about 50 mol % nu-carrageenan.
In another aspect, the galactan comprises agar.
In yet another aspect, the sulfohydrolase comprises 6-O-sulfohydrolase.
In still another aspect, the sulfohydrolase is capable of converting nu-carrageenan into iota-carrageenan.
In another aspect, the sulfohydrolase is capable of converting mu-carrageenan into kappa-carrageenan.
In another aspect, the sulfohydrolase is capable of removing 6-O-sulfate to induce anhydrobridge formation.
In another aspect, the fractionation comprises ammonium sulfate fractionation.
In still another aspect, the method further comprises subjecting at least one of the phenyl sepharose fractions to DEAE sepharose chromatography to obtain DEAE sepharose fractions. At least one of the DEAE sepharose fractions may be subjected to heparin sepharose chromatography.
In another aspect, the incubation is at a temperature of about 0 to 60xc2x0 C.
In still another aspect, the incubation is at a pH of about 5.5 to 9.5.
In yet another aspect, the at least one sulfohydrolase to be added to the reaction mixture is contained within a solution having a concentration of the at least one sulfohydrolase of at least about 2 to 85 xcexcg/ml.
In another aspect, the sulfated compound is at a concentration of about 0.012 to 2% (w/v) in the reaction mixture.
In yet another aspect, the modified compound comprises iota-carrageenan.
In still another aspect, the modified compound comprises kappa-carrageenan.
In another aspect, the enzymatically modified compound comprises precursor enriched kappa-carrageenan.
In another aspect, the enzymatically modified compound comprises precursor enriched iota-carrageenan.
In another aspect, the removal of sulfate comprises removing 6-sulfate group from a galactan.
In still another aspect, the removal of sulfate comprises converting nu-carrageenan to iota-carrageenan to form a product that does not gel under conditions wherein the product has a concentration of about 0.7% (w/v) in an aqueous solution having 0.1 M of potassium at a temperature of 40xc2x0 C. and a pH of 7.
In another aspect, the first sulfohydrolase removes sulfate randomly.
In yet another aspect, the second sulfohydrolase removes sulfate processively.
In another aspect, the seaweed comprises Spinosum.
In still another aspect, the seaweed is freeze-dried and milled prior to being dispersed in the salt solution.
In yet another aspect, the salt solution further comprises KCl. The KCl may have a concentration of about 1 to 1.5 M.
In another aspect, the dispersion is allowed to sit for at least about 18 hours prior to filtering.
In another aspect, the concentrating is by a factor of about 2 to 3.
In still another aspect, the adjusting of the pH of the liquid is by adding base.
In another aspect, the precipitating is by adding isopropyl alcohol.
In yet another aspect, the dialyzing is conducted until the conductivity of the water is stabilized at less than about 2 xcexcS/cm.
In another aspect, after the precipitating of the dialyzed one of nu- and mu-carrageenan, the one of nu- and mu-carrageenan is freeze-dried and milled.
In another aspect, the precipitated dialyzed one of nu- and mu-carrageenan has a one of nu- and mu-content of about 18 to 35 mol %.
In another aspect, the precipitated dialyzed one of nu- and mu-carrageenan has a molecular weight of about 700 to 800 kDa.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the various embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
All percent measurements in this application, unless otherwise stated, are measured by weight/volume based upon grams per milliliter. Thus, for example, 30% represents 30 grams out of every 100 milliliters of the sample.
Unless otherwise stated, a reference to a compound or component, includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.
Before further discussion, a definition of the following terms will aid in the understanding of the present invention.
xe2x80x9cGalactanxe2x80x9d: a polysaccharide composed of galactose units and additional units; such as agars and carrageenans. Galactans have at least 50 mol % of galactose units.
xe2x80x9cHydrocolloidxe2x80x9d: a hydrophilic polysaccharide or derivative, e.g., plant polysaccharide, that swells to produce a viscous dispersion or solution when added to water.
xe2x80x9cHybridization conditionsxe2x80x9d: nucleic acid of interest is transferred onto Nylon membranes, available from Amersham Pharmacia Biotech AB, as described in SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety. The membranes are hybridized overnight in 6xc3x97SSC, 5xc3x97Denhar""t, 0.1% SDS and 100 xcexcg/ml of salmon sperm DNA at 42xc2x0 C. In this regard, SSC is an aqueous solution of 3 M sodium chloride and 0.3 M sodium citrate made in accordance with the procedure described in SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety. After hybridization, filters were washed at 42xc2x0 C. for 15 minutes in the following solutions, 2xc3x97SSC, 0.1% SDS; 1xc3x97SSC, 0.1% SDS and exposed to photostimulated screen, available from Molecular Dynamics, Uppsala, Sweden, scanned using Storm, also available from Molecular Dynamics, Uppsala, Sweden.
To determine the percent identity or percent homology of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=# of identical positions/total # of positions (e.g., overlapping positions).times.100). Preferably, the two sequences are the same length.
The determination of percent homology between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of KARLIN et al., Proc. Natl. Acad. Sci. USA, 87, pp. 2264-2268 (1990), the disclosure of which is herein incorporated by reference in its entirety, modified as in KARLIN et al., Proc. Natl . Acad. Sci. USA, 90, pp. 5873-5877 (1993), the disclosure of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of ALTSCHUL et al., J. Mol. Biol., 215, pp. 403-410 (1990), the disclosure of which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to sulfohydrolase nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to sulfohydrolase protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in ALTSCHUL et al., Nucleic Acids Res., 25, pp. 3389-3402 (1997), the disclosure of which is herein incorporated by reference in its entirety. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of MYERS et al., CABIOS, 4, pp. 11-17 (1988), the disclosure of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
As an overview, the present invention relates to the isolation and purification of sulfohydrolases. For instance, the sulfohydrolases may be purified from seaweed. The sulfohydrolases are preferably those which are able to remove sulfate from sulfated compounds, such as carrageenan. The sulfohydrolases may also be able to form 3,6-anhydro bridges on the sulfated compounds, e.g., carrageenan. The present invention also relates to methods of enzymatic modification of sulfated compounds. The present invention is further directed to modified sulfated compounds, such as modified carrageenans, such as nu-carrageenan which has been modified to at least have portions which correspond to iota-carrageenan, or such as mu-carrageenan which has been modified to at least have portions which correspond to kappa-carrageenan. The present invention is also directed to methods of extracting carrageenan from seaweed.
Given the guidance in the present application and the current state of the art of: (1) protein purification and (2) partial amino acid sequence determination; (3) construction and use of probes to locate corresponding DNA sequencing; and (4) cloning and sequencing, the discussion which follows makes it possible to (1) isolate the sulfohydrolases; (2) identify their amino acid sequences; (3) construct probes for the corresponding DNA sequences; and (4) identify, isolate, purify, and determine the DNA sequences encoding the enzymes. See, for example, SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety.
The compounds to be enzymatically modified by the present invention include sulfated compounds. The sulfated compounds include galactans, glycosaminoglycans, and fucans.
Fucans are highly heterogeneous sulfated polysaccharides composed of different sugar residues, such as fucose, galactose, mannose, and uronic acid. The number of fucan groups and their compositional patterns differ considerably. Fucans are produced in brown algae and in Echinoderm. In this regard, fucans may be from the matricial phase of the cell walls of brown algae, which primarily contain L-fucose. L-fucose residues are often linked by alpha(1xe2x86x923) linkages and sulfated on the C4 position. Enzymatic modification of fucan may affect the anti-coagulating properties of fucans.
Glycosaminoglycans (GAGs) is a family of sulfated polysaccharides from the extracellular matrix of animals, which encompasses, heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate. GAGs consist of disaccharide repeating units containing hexuronic acid and hexosamine. Heparin and heparin sulfate are variably sulfated glucosaminoglycans that consists primarily of alternating alpha(1xe2x86x924)-linked residues of D-iduronate-2-sulfate or D-glucuronate-2-sulfate and N-sulfo-D-glucosamine-6-sulfate. Enzymatic modification of heparin may affect the anti-clotting properties of heparin. Chondroitin sulfate and dermatan sulfate consist primarily of alternating alpha(1xe2x86x924)-linked residues of D-iduronate or D-glucuronate and N-sulfo-D-galactosamine-6-sulfate. Usually the C2 or C4 position of the N-acetylgalactosamine is sulfated and the C2 position of iduronic acid in dermatan sulfate is also frequently sulfated. Keratan sulfate disaccharide consists of galactose and N-acetylglucosamine. The C6 position of either the galactose or the N-acetylglucosamine can be sulfated.
Galactans may be obtained from many different sources. For example, galactans may be obtained from seaweed. Examples of galactans include carrageenan and agar.
As an example, the extraction of agar from seaweed is known. For instance, the extraction of agar from seaweeds is described in SELBY et al., xe2x80x9cAgarxe2x80x9d, Industrial Gums: Polysaccharides and their Derivatives, 3rd ed., (1993), the disclosure of which is herein incorporated by reference in its entirety.
The extraction of carrageenan from seaweeds is known. For instance, the extraction of carrageenan from seaweeds is described in THERKELSEN, xe2x80x9cCarrageenanxe2x80x9d, Industrial Gums: Polysaccharides and their Derivatives, 3rd ed., (1993), the disclosure of which is herein incorporated by reference in its entirety.
The present invention is also directed to a method for extracting higher than natural levels of nu- and mu-carrageenan with high molecular weight, i.e., above about 500 kDa. Depending upon the carrageenan to be extracted, the source for carrageenan may be a seaweed, e.g., Spinosum, Cottonii, and Gigartina radula. 
The seaweed may be freeze-dried, milled, and dispersed in an aqueous KCl solution having a concentration of preferably about 0.05 to 0.3 M, more preferably about 0.1 to 0.15 M, and most preferably about 0.125 M KCl. The dispersion also contains K2CO3 at a concentration of preferably about 0.0005 to 0.003 M, more preferably about 0.001 to 0.0015 M, and most preferably about 0.00125 M to maintain an alkaline environment, i.e., a pH of preferably about 7 to 9, more preferably about 7.5 to 8.5, and most preferably about 7.8 to 8.3. The corresponding calcium salts, i.e., chlorides and carbonates, may also be used, but since the solubility of these is relatively poor, potassium salts are preferred.
The dispersion is allowed to sit for a period of time, preferably about 1 to 72 hours, more preferably about 16 to 32 hours, and most preferably about 24 hours, at a temperature of about 5 to 55xc2x0 C., more preferably about 20 to 50xc2x0 C., and most preferably about room temperature.
After this period of time, the dispersion is filtered. For example, filtration may be conducted with a pressurized kieselguhr-filter.
After filtration, the liquid is preferably ultrafiltered and/or diafiltered, e.g., with an MWCO-membrane of 30 kDa, to remove excess salts. Late in the ultrafiltration, washing of the extract is preferably done by addition of tap water at room temperature. The washing is preferably done until conductivity is below 2 mS/cm and stable. The liquid is preferably concentrated on the ultrafiltration to 25 to 30%. The pH is preferably adjusted to about 8 to 8.5 with, e.g., 0.1 M NaOH.
The liquid is then evaporated to preferably about 10 to 90%, more preferably about 30 to 70%, and most preferably about 50%. For instance, the liquid may be evaporated on a vacuum evaporator.
After evaporation, the pH is adjusted to preferably about 7 to 9, more preferably about 7.5 to 8.5, and most preferably about 8. For instance, the pH may be adjusted to about 8 by adding 1 M NaOH. The carrageenan is then precipitated using isopropyl alcohol (IPA), e.g., 100% (v/v) IPA, available from BP Chemicals Ltd., UK and freeze-dried.
The resulting extract has an enriched nu- or mu-content of preferably about 15 to 40 mol %, more preferably about 17 to 30 mol %, and most preferably about 19 to 24 mol %. The resulting extract also has a high molecular weight of preferably about 500 to 1100 kDa, more preferably about 600 to 900 kDa, and most preferably about 700 to 800 kDa.
The sulfohydrolases of the present invention may be isolated from seaweeds producing sulfated galactans, such as the families of Solieriaceae (such as Eucheuma, Kappaphycus), Gigartinaceae (such as Chondrus, Gigartina), Furcellariaceae, Hypneaceae, Phyllophoraceae, Cystocloniaceae (such as Calliblepharis), and Bangiaceae (such as Porphyra). For example, a non-exhaustive list of seaweed sources and potential seaweed sources for sulfohydrolases include Eucheuma spinosum, Eucheuma cottonii (=Kappaphycus alvarezii), Eucheuma denticulatum, Chondrus crispus, Calliblepharis jubata, Gigartina radula, Gigartina stellata, and Porphyra umbilicalis. 
The following paragraphs describe techniques for isolating the enzyme. From the guidance and examples of purification techniques described in the present application, a skilled artisan would be able to develop additional techniques for isolating the sulfohydrolases.
To isolate sulfohydrolases in accordance with the present invention, it is often preferred that potential enzyme sources, e.g., seaweeds, be screened for enzyme activity to ensure that an enzyme of interest is present in the potential enzyme source. As discussed in more detail below, the screening typically involves obtaining a crude extract from the potential enzyme source. The crude extract would then be used to treat a variety of substrates under a variety of conditions to generally determine the activity of any enzymes present in the crude extract. With this information, a skilled artisan would be able to generally determine whether it might be worthwhile to subject the potential enzyme source to more thorough purification, as discussed below, and to generally determine conditions under which an enzyme is stable and active.
As an example of a crude extraction technique, to isolate enzymes from C. crispus, gametophyte plants of C. crispus may be frozen in liquid nitrogen and ground to a powder. Although the powder may be used immediately, to maintain the integrity of enzymes in the powder, the powder may be kept at a low temperature, such as xe2x88x9220xc2x0 C. to xe2x88x9280xc2x0 C. until further use.
This powder may then be extracted with a buffer, e.g., 4 volumes of 50 mM Tris-HCl buffer (pH 9.5) containing 10 mM 2-mercaptoethanol and 500 mM KCl. After centrifugation, the supernatant may then be fractionated with cold (e.g., xe2x88x9220xc2x0 C.) acetone, e.g., in a two step process: (1) from 0 to 30% saturation; and (2) from 30 to 60% saturation of acetone. After each fractionation step, a centrifugation is preferably performed, e.g., at 4xc2x0 C. for 30 min. at 10,000xc3x97g. The pellets may be dissolved in a buffer, e.g., 2 ml of 50 mM Tris-HCl buffer (pH 7.1) containing 10 mM 2-mercaptoethanol. The pellets as well as an aliquot of the supernatants may then be dialyzed, e.g., overnight at 4xc2x0 C. against 3xc3x973 liters of 50 mM Tris-HCl buffer (pH 7.1) containing 10 mM 2-mercaptoethanol using a Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA. The pellets and the supernatants may be used to search for sulfohydrolase activity.
As another example of a crude extraction technique, to isolate enzyme from E. cottonii (=Kappaphycus alvarezii), Cottonii may be frozen in liquid nitrogen. The cottonii may be used immediately or kept at low temperature, e.g., xe2x88x9280xc2x0 C., until further use. Prior to extraction the Cottonii may be ground to a fine powder.
All of the following steps may be performed at 4xc2x0 C., unless otherwise noted. The powder may then be subjected to extraction using a buffer, e.g., 50 mM Tris-HCl (pH 9.5)+500 mM KCl and 10 mM 2-mercaptoethanol. The resulting suspension may be stirred, e.g., overnight, and then centrifuged, e.g., at 10,000xc3x97g for 75 min. using a Beckman J2-21, Rotor JA20 available from Beckman Instruments, Inc., Fullerton, Calif., USA. The supernatant may be fractionated with cold acetone as described previously. After centrifugation, supernatant and pellets are collected. Pellets, as well as an aliquot of the supernatant may then be dialyzed, e.g., using an MWCO 6-8000 or 3500 dialysis membrane available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA, for 20 hours against 4xc3x971 liter of 50 mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol. After dialysis, the supernatant and the redissolved pellets may be used to search for sulfohydrolase.
Once a crude extract is obtained, to determine whether an extract has any sulfohydrolase activity, several assays are possible. For instance, sulfohydrolases remove sulfate groups from carrageenan such that measuring the level of free sulfate is an indication of sulfohydrolase activity. 6-O-sulfohydrolases, which are able to remove 6-O-sulfate, also tend to cause the formation of 3,6-anhydrogalactose bridges and to induce, therefore, an increase of viscosity. Therefore, measuring the level of 3,6-anhydrogalactose bridges is another measure of sulfohydrolase activity. Additionally, 6-O-sulfohydrolase activity corresponds with an increase in viscosity such that viscosity may be measured to indicate sulfohydrolase activity.
Regarding the measuring of free sulfate, the activity of the sulfohydrolases on carrageenans is measured in the present application by a new assay for measuring the free sulfate. Several assays for the determination of free sulfate have been described in the literature, but these assays are either time consuming or not sensitive enough. See WONG et al., xe2x80x9cSulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)xe2x80x9d, Plant Physiology, Vol. 61, pp. 663-666 (1978); REES, xe2x80x9cEnzymatic Desulphation of Porphyranxe2x80x9d, Biochem. J., 80, pp. 449-453 (1961); and ZINOUN et al., xe2x80x9cEvidence of Sulfohydrolase Activity in the Red Alga Calliblepharis jubataxe2x80x9d, Botanica Marina, Vol. 40, pp. 49-53 (1997), the disclosures of which are herein incorporated by reference in their entireties.
The new assay for determining free sulfate levels is based on the determination of the free sulfate by high performance anion exchange chromatography using auto suppressed conductivity detection within 8 minutes. Besides being fast and fully automated, this method has the advantage of being reproducible and sensitive since as few as 10 ppm of free sulfate can be easily detected.
For instance, sulfohydrolase activity may be assayed by measuring the amount of sulfate released upon incubation of the enzyme extract on carrageenan. The reaction mixture may contain 100 xcexcl of enzyme sample in 50 mM Tris-HCl (pH 7.1)/10 mM 2-mercaptoethanol and 100 xcexcl of 1.4% (w/v) carrageenan. In this regard, the carrageenan is either in the same buffer or in MilliQ water, available from Millipore Corporation, Bedford, Mass., USA. It should be noted that the enzyme sample may be obtained by any appropriate technique, such as the crude extraction techniques described above. The sample, however, may be any other sample for which a measure of the activity is desired. Unless otherwise noted, a reference mixture is made using enzyme extract boiled for 10 minutes prior to use. After 6 to 15 hours incubation at 48xc2x0 C., carrageenan is removed from the reaction mixture by centrifugation at 3320xc3x97g for 1 hr at 30xc2x0 C. in a Microcon-10 unit, available from Amicon Bioseparations, Millipore Corporation, Bedford, Mass., USA.
The amount of free sulfate present in the filtrate from the Microcon-10 unit is then analyzed by HPAEC (high performance anion exchange chromatography) using a Dionex DX 500 chromatography system equipped with a GP40 gradient pump and an ED40 electrochemical detector, all available from Dionex Corporation, Sunnyvale, Calif., USA. The column, an IonPac AS12 A anion exchange column (4xc3x97200 mm, also available from Dionex Corporation) was mounted on an AG12 Guard column (4xc3x9750 mm, also available from Dionex Corporation). The eluent is 9.5 mM Na2CO3/0.5 mM NaHCO3 at a flow rate of 1.5 ml/min. Detection of anion is performed by ASRS conductivity using an anion self regenerating suppressor ASRS-1 (4 mm, also available from Dionex Corporation) with an SRS (Self Regenerating Suppressor) current of 50 mA.
Regarding measuring 3,6 anhydrogalactose bridges, another assay may be used. The amount of 3,6 anhydrogalactose bridge produced during the desulfatation reaction is measured in this application using the technique described in JOL et al., xe2x80x9cA Novel High-Performance Anion-Exchange Chromatographic Method for the Analysis of Carrageenans and Agars Containing 3,6-Anhydrogalactosexe2x80x9d, Anallyical Biochemstry 268, pp. 213-222 (1999), the disclosure of which is herein incorporated by reference in its entirety.
Regarding measuring viscosity levels, viscometric measurements may be carried out on the reaction mixtures. The viscosity of the reaction mixture is directly measured using a programmable Brookfield rheometer model DV III, available from Brookfield Engineering Laboratories, Stoughton, Mass., USA, thermostated at 48xc2x0 C. Measurements are performed with a CP52 spindle for 10 minutes using a shear rate of 120 rpm.
Once a sample has been identified as a potential source for enzymes, e.g., by the above described screening, the sulfohydrolases may be purified to homogeneity in accordance with the present invention. From the guidance and purification techniques described in the present application, a skilled artisan would be able to develop additional purification techniques.
A typical example of the purification of the enzymes from C. crispus is shown and discussed on the following pages; all steps are performed at 4xc2x0 C. unless otherwise noted: 
As noted above, all steps of fractionation and purification in the above purification process are performed at 4xc2x0 C. unless otherwise noted.
Gametophyte plants of Chondrus crispus may be frozen in liquid nitrogen and ground. For example, the frozen C. crispus may be automatically ground to pieces which are less than 1 mm long by using a Forplex miller, available from Forplex Industrie, Boulogne Billancourt, France. This xe2x80x9cpowderxe2x80x9d may be used immediately or may be kept at low temperature, e.g., xe2x88x9220xc2x0 C. to xe2x88x9280xc2x0 C., until further use.
The ground C. crispus may then be subjected to extraction. For example, this frozen ground C. crispus in the amount of 650 g may be allowed to thaw overnight in 1.5 volumes (v/w) of 4xc2x0 C. extracting buffer (50 mM Tris-HCl, pH 9.5/500 mM KCl/10 mM 2-Mercaptoethanol). In other words, the 650 g of C. crispus may be allowed to thaw in 975 ml of the buffer. The suspension may be stirred overnight and then centrifuged, e.g., at 10,000xc3x97g for 75 min.
The supernatant from extraction may then be subjected to fractionation. For instance, the supernatant may be brought to 30% (NH4)2SO4 saturation (16.4 g ammonium sulfate/100 ml of sample) by adding ammonium sulfate. In this regard, this percentage refers to the percent of saturation in ammonium sulfate which ammonium sulfate solutions are saturated at 3.9 M at 0xc2x0 C. When all the ammonium sulfate is dissolved, the mixture may be allowed to stand for about 30 min. and then centrifuged, e.g., at 24,700xc3x97g for 60 min.
Sulfohydrolase activity usually cannot be detected in the crude extract and sometimes not in the ammonium sulfate supernatant. This property may be the result of interferences with polysaccharides or proteins that are removed during the phenyl sepharose chromatography which is described below. An important purpose of the phenyl sepharose chromatography is to remove phycoerytrin, a hydrophilic protein, which constitutes the major component in the Chondrus extract.
After centrifugation of the ammonium sulfate mixture, the supernatant may be collected and loaded on a phenyl sepharose column, e.g., a Phenyl Sepharose 6 fast flow (2.0xc3x9724 cm) column, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, previously equilibrated in 50 mM Tris-HCl buffer (pH 8.7), 30% (NH4)2SO4 saturation, 500 mM KCl and 10 mM 2-Mercaptoethanol (buffer D). The column may be washed with this buffer up to negligible absorbance at 280 nm in the effluent. The bound proteins may then be eluted with a linear decreasing gradient of (NH4)2SO4 made of 360 ml of buffer D and 360 ml of the same buffer without (NH4)2SO4 (buffer C). At the end of the gradient, phycoerythrin as well as some other proteins are often eluted with buffer C alone. As an example, the elution may be conducted under the following conditions:
Buffer D: 50 mM Tris-HCl buffer, pH 8.7, containing 30% of saturation of (NH4)2SO4, 500 mM KCl, and 10 mM 2-mercaptoethanol.
Buffer C: 50 mM Tris-HCl buffer, pH 8.7, containing 500 mM KCl, and 10 mM 2-mercaptoethanol.
Flow Rate: 1 ml/min
Fraction Size: 8 ml
The above described extraction and phenyl sepharose chromatography are preferably performed at least twice in order to have enough material for subsequent steps.
Fractions of interest, i.e., fractions that are later found to have sulfohydrolase activity, from the phenyl sepharose chromatography may then be dialyzed. For instance, active fractions may be pooled and dialyzed for 36 hr against 5xc3x9710 1 of 50 mM Tris-HCl buffer (pH 7.1) containing 10 mM 2-Mercaptoethanol (buffer A), using a Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA. After dialysis, the pH and the conductivity of the sample may be checked and adjusted to those of buffer A (i.e., pH 7.1 and conductivity of about 1.7 mS/cm) if necessary.
After dialysis, the sample may then be subjected to DEAE Sepharose chromatography. For instance, the sample may be applied at a flow rate of 1 ml/min to a DEAE Sepharose column (2.0xc3x9722 cm), available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, previously equilibrated with buffer A. The column may be washed with buffer A until the absorption at 280 nm is negligible. Then the adsorbed proteins may be eluted, e.g., at a flow rate of 1 ml/min with a linear NaCl (from 0 to 1 M) gradient in buffer A. As an example, the elution may be performed as follows:
Buffer A: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM 2-mercaptoethanol
Buffer B: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM 2-mercaptoethanol+1 M NaCl
Fraction Size: 7.5 ml
Flow Rate: 1 ml/min
SDS PAGE analysis of DEAE Sepharose chromatography fractions eluting between 650 and 800 mM NaCl normally reveal the presence of one single band at 34.9 kDa. This band corresponds to an enzyme denoted sulfohydrolase II which is discussed in more detail below.
In addition to sulfohydrolase II, sulfohydrolase I elutes between 300 and 600 mM NaCl during the DEAE chromatography. These fractions, however, usually include impurities as well. To isolate sulfohydrolase I, further purification, such as dialysis and semi-affinity and cation-exchange chromatography, e.g., heparin chromatography, is required.
As an example of heparin chromatography, half of a fraction eluting with 580 mM NaCl may be dialyzed against buffer A using a Spectra/por membrane MWCO 6-8000 or 3500, available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA. The fraction may then be loaded (flow rate 1.5 ml/min) on top of a HiTrap heparin column, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, previously equilibrated with buffer A. The column may then be washed with this buffer at a flow rate of 1.5 ml/min until the absorbance at 280 nm is negligible. Bound proteins may then be eluted using a three step increasing NaCl gradient (from 0 to 1200 mM). In particular, elution may be conducted using the following gradient:
Buffer A: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM 2-mercaptoethanol
Buffer Bxe2x80x2: 50 mM Tris-HCl buffer, pH 7.1 and 10 mM 2-mercaptoethanol+1.2 M NaCl
Fraction size: 1.5 ml
Flow rate: 1.5 ml/min
SDS-PAGE analysis of fractions eluting with 1200 mM NaCl should reveal a single faint band characterized by a molecular weight of 62.1 kDa. This band corresponds to an enzyme denoted sulfohydrolase I.
The above-described purification procedure preferably starts with 2xc3x97600-700 g of fresh seaweed, such as the above-noted 650 g. With this amount of material, both enzymes separate well after DEAE chromatography, but the sulfohydrolase I is still not pure. The above-described chromatography on semi-affinity and cation exchange chromatography is necessary to purify this enzyme to homogeneity.
Cottonii sulfohydrolases may also be purified using a procedure which is similar to the above procedure for purifying C. crispus sulfohydrolases I and II.
For instance, Cottonii may be frozen in liquid nitrogen and kept at xe2x88x9280xc2x0 C. until further use. Prior to extraction the frozen Cottonii may be ground, e.g., manually to a fine powder in a mortar, using liquid nitrogen which was poured with a baker into the mortar to keep the Cottonii frozen during the grinding.
To partially purify the Cottonii sulfohydrolase, the following steps may be performed at 4xc2x0 C. Ground Cottonii may be extracted in buffer, e.g., 25 g of ground Cottonii in 40 ml of 50 mM Tris-HCl (pH 9.5)+500 mM KCl and 10 mM 2-mercaptoethanol. The resulting suspension may be stirred, e.g., with a magnetic stirrer overnight, and then centrifuged, e.g., at 10,000xc3x97g for 75 min using a Beckman J2-21, Rotor JA20 available from Beckman Instruments, Inc., Fullerton, Calif., USA. The supernatant may be dialyzed, e.g., using a MWCO 6-8000 or 3500 dialysis membrane available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA, for 20 hours against 4xc3x971 liter of 50 mM Tris-HCl (pH 8.5)+10 mM 2-mercaptoethanol. After dialysis, the supernatant may be loaded on a heparin column, e.g., at a flow rate of 1 ml/min on top of a Heparin agarose type II-S column (27.5xc3x971 cm column, 10xc3x970.5 cm gel size), available from Millipore, Stonehouse, England, with the heparin type II-S agarose gel being available from Sigma Chemical, St. Louis, Mo., USA, previously equilibrated in the same buffer. The column may be washed with this buffer until the absorption at 280 nm is negligible. Then, the adsorbed proteins may be eluted, e.g., for 40 minutes with a linear NaCl (from 0 to 1 M) gradient. In particular, elution may be conducted using the following gradient:
Buffer A: 50 mM Tris-HCl (pH 8.5)+10 mM 2-mercaptoethanol
Buffer B: 50 mM Tris-HCl (pH 8.5)+10 mM 2-mercaptoethanol+1 M NaCl
Fraction size: 2 ml
Flow rate: 1 ml/min
Although most of the proteins in the extract typically do not bind to the heparin agarose, the sulfohydrolase binds to the gel as most of the activity, in terms of sulfate released and/or viscosity increase, is found in later fractions. The fractions from the heparin chromatography may be concentrated, e.g., by about 13xc3x97by centrifugation at 3320xc3x97g for 1 hr at 4xc2x0 C. in a Microcon-10 unit, available from Amicon Bioseparations, Millipore Corporation, Bedford, Mass., USA, prior to SDS-PAGE analysis and silver nitrate staining, according to MERRIL et al., xe2x80x9cUltrasensitive Stain for Proteins on Polyacrylamide Gels Shows Regional Variation in Cerebrospinal Fluid Proteinsxe2x80x9d, Science, 211, pp. 1437-1438 (1981), the disclosure of which is herein incorporated by reference in its entirety.
The different active fractions are all characterized by the presence of two common proteins: one which is characterized by a molecular weight of about 65 kDa and another which is characterized by a molecular weight of about 55 kDa.
In accordance with the present invention, based on total weight of protein in the sample, sulfohydrolases may have high purity. The sulfohydrolase is preferably purified to at least about 40 wt %, more preferably at least about 50 wt %, even more preferably at least about 60 wt %, even more preferably at least about 70 wt %, even more preferably at least about 80 wt %, even more preferably at least about 90 wt %, even more preferably at least about 95 wt %, even more preferably at least about 99 wt %, and most preferably 100 wt %, based on total weight of protein. As discussed in more detail below, this high level of purity allows tailoring of the properties of the sulfated compounds to be modified.
Once the sulfohydrolases have been purified, the amino acid sequences may be determined. The peptide sequence of these proteins may be determined by conventional techniques, such as Edman""s degradation.
In order to determine the N-terminal amino acid sequence of both sulfohydrolases, the sulfohydrolases were purified generally in accordance with the above-described purification protocol involving extraction, ammonium sulfate fractionation, phenyl sepharose chromatography, DEAE Sepharose chromatography, etc. In particular, fractions 60 and 61 from the DEAE Sepharose chromatography were concentrated 20xc3x97, by centrifugation at 3320xc3x97g for 1 hr at 4xc2x0 C. in a Microcon-10 unit, available from Amicon Bioseparations, Millipore Corporation, Bedford, Mass., USA, prior to SDS PAGE. Fractions 38 to 47 from the DEAE chromatography were pooled and dialyzed for 6 hours at 4xc2x0 C. against 3xc3x973 liters of 50 mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol by using a MWCO 6-8000 or 3500 dialysis membrane available from Spectrum Laboratories, Inc., Rancho Dominguez, Calif., USA. After dialysis, the conductivity of the sample was adjusted to that of the buffer by dilution with milliQ water, available from Millipore Corporation, Bedford, Mass., USA. Half of the sample (57 ml) was then loaded (flow rate 1.5 ml/min) on top of a HiTrap heparin column available from Pharmacia Biotech AB, Uppsala, Sweden, previously equilibrated with 50 mM Tris-HCl (pH 7.1)+10 mM 2-mercaptoethanol. The column was washed with this buffer until the absorbance at 280 nm was negligible. Then, elution of proteins was performed using an increasing gradient (from 0 to 1.2 M) of NaCl. Fractions 24, 25, 26, and 28, which constitute the peak of elution for absorbance at 280 nm, were concentrated about 13xc3x97, by centrifugation at 3320xc3x97g for 1 hr at 4xc2x0 C. in a Microcon-10 unit, available from Amicon Bioseparations, Millipore Corporation, Bedford, Mass., USA, before being loaded on a SDS gel for SDS PAGE.
Accordingly, two fractions from the DEAE chromatography (fractions 60 and 61) as well as four fractions from HiTrap Heparin chromatography (fractions 24, 25, 26, and 28), were subjected to SDS PAGE (12% total monomer (acrylamide+N,Nxe2x80x2-methylenebisacrylamide) in grams per 100 ml gels) in accordance with the technique described in LAEMMLI, Nature, 227, pp. 680-685 (1970), the disclosure of which is herein incorporated by reference in its entirety, to separate the enzymes.
After SDS PAGE, the proteins were transferred onto a xe2x80x9cHybond-Pxe2x80x9d membrane (polyvinylidene difluoride (PVDF) membrane), available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. The transfer was performed for 2 hrs at 250 mA in 50 mM Tris-HCl buffer (pH 8.25) containing 50 mM borate. After transfer, the proteins were then fixed for few seconds in 100% (w/v) methanol and the membrane was then stained for 1 minute with a solution of 0.1% (w/v) Coomassie Blue R250, available from Bio-Rad Laboratories, Hercules, Calif., USA, in 50% (v/v) methanol and 10% (v/v) acetic acid. Destaining is carried out by soaking the membrane for 4-5 minutes in a 50% (v/v) methanol and 10% (v/v) acetic acid solution. After rinsing for 1-2 minutes with MilliQ water, available from Millipore Corporation, Bedford, Mass., USA, the bands corresponding to sulfohydrolases I and II were cut and sent to the Institute Pasteur, Paris, France, for N-terminal amino acid sequence determination by Edman""s degradation. From this experiment, it appears that sulfohydrolase II has a blocked amino-terminal end and, therefore, its amino-terminal sequence could not be determined. The Edman""s degradation of the sulfohydrolase I resulted in a determination that the N-terminal had the amino acid sequence of SEQ ID NO: 1.
A similar strategy was used to determine the amino acid sequence of some internal peptides. In this case, however, SDS PAGE (12% (total monomer (acrylamide+N,Nxe2x80x2-methylenebisacrylamide) in grams per 100 ml gel) gels) in accordance with the technique described in LAEMMLI, Nature, 227, pp. 680-685 (1970), the disclosure of which is herein incorporated by reference in its entirety, was not followed by a transfer. In particular, immediately after electrophoresis, the gel was stained overnight with a 0.0033% (w/v) amido black solution in 50% (v/v) methanol and 10% (v/v) acetic acid. The gel was then briefly rinsed (4xc3x9710 min.) with MilliQ water, available from Millipore Corporation, Bedford, Mass., USA, in order to remove traces of methanol and acetic acid. Then, the bands corresponding to sulfohydrolases I and II were excised and dried for about 30 min. at 30xc2x0 C. in a centrifuged evaporator RC 10.10, Jouan, France, equipped with a refrigerated trap RCT 90, Jouan, France, before being sent to the Institute Pasteur, Paris, France, for proteolytic digestions and microsequence analysis, as discussed below.
Slices of acrylamide, which contained about 15-30 xcexcg of sulfohydrolase I, were submitted to proteolysis with 0.4 xcexcg endoprotease-LysC in 350 xcexcl of 0.05 Tris-HCl (pH 8.6)/0.03% (w/v) SDS for 18 h at 37xc2x0 C. The resulting peptides were then submitted to reverse-phase HPLC on a 250 mmxc3x972.1 mm DEAE C18 column, available from CIL CLUZEAU, Paris, France. Elution, carried out with an acetonitrile gradient from 2 to 45% (v/v) in 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.2 ml/min, allowed many peptides to be separated. Among them, peptides 11, 12, 18, 22, and 25 were significant and were collected for sequencing. These peptides were analyzed at the Institute Pasteur, Paris, France, by Edman""s degradation using an Applied Biosystems 473A automated gas phase amino acid sequencer, available from PE Applied Biosystems, Foster City, Calif., USA, in accordance with standard procedures. As a result of this analysis, sulfohydrolase I was determined to include SEQ ID NOS: 2-5.
Acrylamide slices containing about 10 xcexcg of SDS PAGE purified sulfohydrolase II were subjected to proteolysis in 600 xcexcl of 0.05 M Tris-HCl (pH 8.6)/0.01% (v/v) xe2x80x9cTween 20xe2x80x9d polyoxyethylene sorbitan monolaurate, available from USB, Cleveland, Ohio, USA, and 4% (w/v) of bovine trypsin. Tryptic digestion was carried out for 18 h at 37xc2x0 C. The resulting tryptic peptides were purified by reverse-phase HPLC on a 250 mmxc3x972.1 mm DEAE C18 column, available from CIL CLUZEAU, Paris, France. Elution was performed with an acetonitrile gradient from 2 to 45% (v/v) for 60 minutes in 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.2 ml/min. Among the isolated peptides, peptides 13, 14, 24, and 25 were collected because they were the most significant, and were analyzed at the Institute Pasteur, Paris, France, by Edman""s degradation using an Applied Biosystems 473A automated gas phase amino acid sequencer, available from PE Applied Biosystems, Foster City, Calif., USA, in accordance with standard procedures. As a result of this analysis, sulfohydrolase I was determined to include SEQ ID NOS: 6-11.
On the basis of the above indicated amino acid sequences, sequence probing processes were carried out for the corresponding cDNA to determine the nucleotide sequence for sulfohydrolase I and II. As described in more detail below, mRNA was isolated from C. crispus, from the mRNA a cDNA library was synthesized, the cDNA library was probed with PCR fragments obtained using oligonucleotides based on the above-described amino acid sequences for sulfohydrolase I and II, and positive cDNAs were sequenced.
Total RNA was prepared from gametophytes of Chondrus crispus as described by APT et al., xe2x80x9cThe Gene Family Encoding the Fucoxanthin Chlorophyll Proteins from the Brown Alga Macrocystis pyriferaxe2x80x9d, Mol. Gen. Genet., 246, pp. 455-464 (1995), the disclosure of which is herein incorporated by reference. Purification of the mRNA was then performed using the PolyA T tract mRNA Isolation system IV kit, available from Promega, Madison, Wis., USA, according to the instructions of the manufacturer.
Once the mRNA was isolated, the cDNA library for C. crispus was constructed. The cDNA synthesis was performed using a lambda ZAP (copyright)II vector cDNA synthesis kit, available from Stratagene, La Jolla, Calif., USA, according to the instructions of the manufacturer. Double stranded cDNA were fractionated through a sepharose CL-2B column, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. The fractions with an average size of 600 to 1500 pb, as estimated on acrylamide gel (5%), were selected for the preparation of the library. Ligation of cDNA into lambda ZapII vector as well as transformation of the host strain XL1-Blue MRFxe2x80x2 were performed according to the instructions of the manufacturer (Stratagene, La Jolla, Calif., USA).
The probe design and screening protocol for screening the cDNA library was as follows. From SEQ ID NOS: 2 and 10, respectively, degenerated oligonucleotides for sulfohydrolase I and II were designed which correspond to SEQ ID NO: 12 for sulfohydrolase I and SEQ ID NOS: 13-14 for sulfohydrolase II. Degenerated oligonucleotide, SEQ ID NO: 12 was used with vector primer SEQ ID NO: 16 (5xe2x80x2AATACGACTCACTATAG3xe2x80x2) to amplify by PCR DNA fragments corresponding to sulfohydrolase I, in the C. crispus cDNA library.
Degenerated oligonucleotides, SEQ ID NOS: 13 and 14 were used with vector primer SEQ ID NO: 15 (5xe2x80x2ATTAACCCTCACTAAAG3xe2x80x2) to amplify by PCR DNA fragments corresponding to sulfohydrolase II in the C. crispus cDNA library as follows. A first PCR using oligonucleotides SEQ ID NO: 14 and SEQ ID NO: 15 gave DNA fragments. Using this first PCR product and oligonucleotides SEQ ID NO: 15 and SEQ ID NO: 13, a DNA fragment corresponding to sulfohydrolase II was amplified.
The cloned PCR fragments corresponding to the sulfohydrolase I gene and the sulfohydrolase II gene were labelled by random priming using the Megaprime kit, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, and 1850 kBq of 32P dCTP, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. The resulting probes were purified using Sephacryl SR 200, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, and used to screen the C. crispus gametophytes cDNA library.
For both sulfohydrolase I and II screenings, plaques were transferred onto Nylon membranes, available from Amersham Pharmacia Biotech AB, as described in SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety. The membranes were hybridized overnight in 6xc3x97SSC, 5xc3x97Denhar""t, 0.1% SDS and 100 xcexcg/ml of salmon sperm DNA at 65xc2x0 C. In this regard, SSC is an aqueous solution of 3 M sodium chloride and 0.3 M sodium citrate made in accordance with the procedure described in SAMBROOK et al., Molecular Cloning A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety. After hybridization, filters were washed 15 minutes in the following solutions, 2xc3x97SSC, 0.1% SDS; 1xc3x97SSC, 0.1% SDS and 0.4xc3x97SSC, 0.1% SDS and exposed to photostimulated screen, available from Molecular Dynamics, Uppsala, Sweden, scanned using Storm, also available from Molecular Dynamics.
For sulfohydrolase I, using the screening conditions described above, the above-described labelled probes for sulfohydrolase I were used in three rounds of screening to screen 300,000 phages from the cDNA library for the presence of sulfohydrolase I cDNA. A total of 408 positive phages (1.4% no %) were positive from which 30 were selected. In a second round, 10 of these positives were screened from which 7 were positive. From these, 6 were found positive in a third round of screening.
For sulfohydrolase II, using the screening conditions described above, the above-described labelled probes for sulfohydrolase II were used in three rounds of screening to screen 600,000 phages from the cDNA library for the presence of sulfohydrolase II cDNA. Twelve positive phages (1/50,000) were found from which 10 were selected. In a second round these positives were screened from which 9 were positive. From these 9, 9 were found positive in a third round of screening. The 5xe2x80x2 and 3xe2x80x2 extremities of the 9 positive cDNAs were sequenced and sequencing revealed that only 4 of the 9 corresponded to the sulfohydrolase II cDNAs. Thus, the sulfohydrolase II gene seems to be weakly expressed in C. crispus gametophyte (1/150,000 cDNAs).
For both sulfohydrolase I and II, in vivo excision was achieved according to the instruction of the manufacturer (packaging kit, available from Stratagene, La Jolla, Calif., USA), and the 5xe2x80x2 and 3xe2x80x2 termini of each cDNA were sequenced, enabling their identification. In particular, the sequences were determined using the Thermo-Sequenase core sequencing kit with 7-deaza-dGTP from Vistra TM. Sequence reactions were run on a Vistra DNA automated sequencer 725, available from Molecular Dynamics, Uppsala, Sweden.
Using the above method, the sequence of the sulfohydrolase I cDNA was determined to be SEQ ID NO 17. In particular, one cDNA was fully sequenced and the sequence termini were determined for the others. Sequencing revealed that they are identical cDNAs, five are full length cDNAs and one is a partial length cDNA.
The sequence for sulfohydrolase I contains the ATG start codon, the signal peptide (possibly for secretion from the ER to the cell wall) with the probable amino acid cleavage between Alanine 20 and Lysine 21, the stop codon and the 3xe2x80x2 UTR (untranslated region).
The above method suggests that the nucleotide sequences for sulfohydrolase II cDNAs are SEQ ID NOS: 18, 19, 20, and 21. In this regard, it is apparent that these 4 cDNAs are different. Three (3) cDNAs are full-length (SEQ ID NOS: 19, 20, and 21) and one may be a partial length cDNA (SEQ ID NO: 18).
From the nucleotide sequences of both sulfohydrolase I and II, the corresponding amino acid sequences were deduced. In particular, the amino acid sequence of sulfohydrolase I was found to be SEQ ID NO: 22. The amino acid sequence of sulfohydrolase II was found to be SEQ ID NO 23.
Regarding the amino acid sequence for sulfohydrolase I, all protein microsequences determined by the Edman""s degradation were found in the sequence. The amino acid sequence of sulfohydrolase I has a 20 amino acid long signal peptide. The cleavage site is between Ala 20 and Lys 21. Therefore, the mature enzyme comprises 594 amino acids and contains one potential N-glycosylation site (NFTI; AA 72-75) and has some similarity with the FAD binding domain.
Sulfohydrolase I shares at its C-terminus, 22% of sequence identity over 300 amino acids with the L-amino acid oxidase of Chlamydomonas reinhardtii, which was described in VALLON et al., xe2x80x9ccDNA Sequence of M(Alpha), the Catalytic Subunit of the Chlamydomonas reinhardtii L-Amino Acid Oxidase (Accession No. U78797): a New Sequence Motif Shared by a Wide Variety of Flavoproteinsxe2x80x9d, Plant Physiol., 115, pp. 1729-1731 (1997), the disclosure of which is herein incorporated by reference in its entirety. In this regard, L-amino-acid oxidases have been found in a variety of organisms. They catalyze the reaction: H2Nxe2x80x94CHRxe2x80x94COOH+O2+H2Oxe2x86x92Oxe2x95x90CRxe2x80x94COOH+NH3+H2O2. In C. reinhardtii, it has been shown that this 65 kDa enzyme is periplasmic, gametespecific and induced by ammonium deprivation.
Sulfohydrolase II is 279 amino acids long and contains one glycosylation site. Analysis of sulfohydrolase II sequences indicated that no significant similarity was found with known sequences in the databases available at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) using BLAST software.
The determination of the amino acid sequences of sulfohydrolase I and II also allows an accurate determination of the molecular weights of these enzymes. In this regard, as estimated by SDS PAGE, the molecular weights of sulfohydrolase I and II are 62.1 and 34.9 kDa, respectively. In the case of the of sulfohydrolase I, this MW is slightly lower than the one deduced from the amino acid sequence which gave a molecular weight of 66.8 kDa. In contrast, relative to the SDS PAGE estimate, sulfohydrolase II has a slightly lower molecular weight of 30.9 kDa when calculated from the amino acid sequence.
From both sequences a theoretical isoelectric point (pI) was calculated using the program Mac Molly translate. Sulfohydrolase I and II are characterized by a pI of 8.4 and 9.3 respectively.
From the amino acid and nucleotide sequences of sulfohydrolase I and II, analogs or homologs of these sulfohydrolases may be detected and isolated using known techniques based upon sequence homology to the sulfohydrolases enzymes disclosed herein. Thus, all or part of the known enzyme coding sequence may be used to construct a probe which will hybridize selectively to identical or highly similar enzyme coding sequences present in genomic or cDNA libraries from a particular source. In particular, nucleotide sequences having a % homology of at least about 25%, more preferably at least about 50%, and most preferably at least about 80%, relative to the specifically identified sequences of the present invention, are in accordance with the present invention. Similarly, amino acid sequences having a % homology of at least 25%, more preferably at least 50%, and most preferably at least 80%, relative to the specifically identified sequences of the present invention, are in accordance with the present invention.
Hybridization techniques include hybridization screening of plated cDNA libraries, and amplification by polymerase chain reaction (PCR) using oligonucleotide primers that correspond to conserved sequence domains. See INNIS et al., PCR Protocols, a Guide to Methods and Applications, Academic Press (1990), the disclosure of which is herein incorporated by reference in its entirety.
Further, references regarding the above techniques include AUSUBEL et al., Current Protocols in Molecular Biology, Green Publishing Company Assoc. and John Wiley Interscience, (1992); and, BERGER et al., Guide to Molecular Cloning Techniques, Academic Press (1987), the disclosures of which are herein incorporated by reference in their entireties.
Homologous sulfohydrolases may be found, for example, by extracting DNA and RNA from the source of interest and conducting Southern hybridization, genomic and cDNA library screening, using a complete or partial sequence from one of the known sulfohydrolases.
For instance, total DNA may be extracted in accordance with the method disclosed in APT et al., xe2x80x9cThe Gene Family Encoding the Fucoxanthin Chlorophyll Proteins from the Brown Alga Macrocystis pyriferaxe2x80x9d, Mol. Gen. Genet., 246, pp. 455-464 (1995), the disclosure of which is herein incorporated by reference. In this regard, it should be noted that it is often not necessary to remove polysaccharides in the manner described by APT et al.
Southern hybridization may be conducted by, for example, by digesting 7 xcexcg of total DNA of Chondrus crispus, Eucheuma cottonii, Eucheuma spinosum, and Gracilaria gracilis for 4 hours at 37xc2x0 C. with 60 U of HindIII, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, and 80 U of EcoRI, available from Biolabs, Beverly, Mass., USA. After precipitation, the digested DNA may be redissolved in 40 xcexcl of 100 mM Tris (pH 7.5)+10 mM EDTA. The fragments may then be fractionated on a 0.8% agarose gel (migration overnight at 35 V) before being transferred under vacuum for 3 hours on a Hybond-N+membrane, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden, using a xe2x80x9cTrans DNA express vacuum blotterxe2x80x9d apparatus, available from Appligene, Gaithersburg, Md., USA, under the conditions given by the manufacturer.
Prehybridation and hybridization may be performed at 60xc2x0 C. for Chondrus crispus and at 42xc2x0 C. for the other algae in the conditions given by SAMBROOK et al., Molecular Cloning: A Laboratory Manual, 2nd ed., CSH Laboratory Press, Cold Spring Harbor, N.Y. (1989), the disclosure of which is herein incorporated by reference in its entirety. After hybridization, the membranes were washed as follows:
For all the algae except Chondrus crispus 
2xc3x97SSC+0.1% SDS for 2xc3x9720 min; and
1xc3x97SSC+0.1% SDS for 2xc3x9715 min.
For Chondrus crispus 
Same as above with an additional wash in 0.5xc3x97SSC+0.1%SDS for 15 min.
Southern hybridizations were conducted by using the partial length cDNA encoding for sulfohydrolase I. After digestion of the cDNA with EcoRI and XhoI, both available from Biolabs, Beverly, Mass., 2 fragments were generated: one of 400 bp and the other one of 1600 bp. This latter one was then collected and labelled using the ECF Random-Prime Labelling and detection System, available from Amersham Pharmacia Biotech AB, Uppsala, Sweden. Labelling, hybridization and detection were performed as recommended by the manufacturer.
Southern hybridization may also be done by using the full length cDNA encoding for the sulfohydrolase II. This Southern hybridization reveals one major band of about 2.5 kB as well as 3 other bands (which size is between 4 an 1.2 kB) in Chondrus crispus. However, even after 60 hours of exposition, 3 faint visible bands could be detected in Eucheuma spinosum and cottonii but nothing could be detected in Gracilaria gracilis. Similar results were obtained using the full length cDNA encoding for sulfohydrolase I as a probe.
The results are summarized below:
All these results indicate therefore that homologous sulfohydrolases I exist in E. spinosum, E. cottonii and in Gigartina skottsbergii and homologous sulfohydrolases II exist in E. spinosum and in E. cottonii, but homology with the respective Chondrus enzymes is low.
Almost pure sulfohydrolase II, which in this case had been purified to 80-90% (w/w) homogeneity, based on the total amount of protein in the sample, was incubated with the iota-precursor giving a gel, whereas the boiled enzyme did not. The enzyme was also incubated in 4 different concentrations with the iota-precursor. Low amounts of enzyme created a soft gel whereas higher amounts gave a stronger gel.
After incubation of nu-carrageenan with sulfohydrolase I, sulfate release was detected but there was no increase in viscosity of the carrageenan sample. In contrast, incubation with sulfohydrolase II resulted in sulfate release and an increase in viscosity. NMR analysis showed that all of the isolated enzyme fractions acted to convert nu-carrageenan to iota-carrageenan.
Because of the observed modifications, the previous theory advanced in U.S. Provisional Application No. 60/133,376 of a sulfohydrolase and an anhydrolase has now been abandoned. This previous theory described the enzymes as possessing two different functions: (1) one enzyme to remove the C-6 sulfate on the nu precursor; and (2) one enzyme to form the 3,6-anhydro-galactose bridge, thereby converting the structure to iota carrageenan. It should be noted, however, that although U.S. Provisional Application No. 60/133,376 apparently failed to accurately describe the mode of action for the enzyme, this provisional application disclosed how to purify and use these enzymes.
While not wishing to be bound by theory, it is now believed that sulfohydrolase I is removing the sulfate groups randomly. In contrast, sulfohydrolase II might act progressively by sliding along the molecule to thereby create stretches of iota, which can aggregate with other similar molecules to create a gel.
When incubating the iota-precursor with first the random enzyme followed by inactivation and thereafter incubation with the processive enzyme, a higher viscosity was obtained when compared to using the processive enzyme alone. It seems that the random enzyme is preparing the polymer in such a way that it is easier for the processive enzyme to slide along the molecule.
Taking into consideration the above, the high purity sulfohydrolases of the present invention allow the tailoring of properties of sulfated compounds.
An advantage of using an enzyme is that it can work at relatively low temperatures, such as 0 to 60xc2x0 C., more preferably 20 to 55xc2x0 C., and most preferably 37 to 48xc2x0 C. Accordingly, the enzyme does not need strong heating to make the carrageenan gel.
The incubation is preferably conducted at a pH of about 5.5 to 9.5, more preferably about 6 to 9.5, and most preferably about 7 to 9. In this regard, incubation with the C. crispus sulfohydrolases is preferably conducted at a pH of about 6.5 to 7.5. Incubation with the Cottonii sulfohydrolase is preferably conducted at a pH of about 8 to 9.5.
The concentration of total proteins of the samples containing the sulfohydrolases is preferably at least about 2 to 85 xcexcg/ml, more preferably about 10 to 80 xcexcg/ml, and most preferably about 25 to 70 xcexcg/ml. In the case of almost pure sulfohydrolase II, the concentration is preferably 1 to 10 xcexcg/ml, more preferably about 2.5 to 10 xcexcg/ml, and most preferably about 2.5 to 5 xcexcg/ml.
The concentration of the substrate, e.g., carrageenan, in the reaction mixture is preferably about 0.012 to 2% (w/v), more preferably about 0.12 to 2% (w/v), and most preferably about 0.7 to 1.75% (w/v).
For incubation with Cottonii extract, the optimal conditions in which the desulfatation reaction should be performed are as follows:
Substrate: mu-carrageenan at a final concentration of about 1.5% (w/v)
Activity /stability as a function of temperature: 9 hours incubation at 48xc2x0 C. releases as much free sulfate as incubation for 16 hours at 37xc2x0 C. or 48 hours at 20xc2x0 C.
Activity/stability as a function of pH: Tris-HCl 100 mM (pH 9.0)+2-mercaptoethanol 10 mM or Bis-tris 100 mM (pH 8.5)+10 mM 2-mercaptoethanol seem to be the most appropriate buffers for the sulfohydrolase of Cottonii
Amount of proteins: 5 xcexcg of total proteins is the minimum amount of material required to get a significant signal on the Dionex.
The purified sulfohydrolases of the present invention are effective in modifying sulfated compounds, e.g., carrageenan. For instance, 1 mg of enzyme extract in accordance with the present invention (in this case 0-30% acetone fraction) is able to release 400 nmoles of sulfate per hour.
Sulfohydrolase I and II may be used separately, or in combination, to modify substrates, and in particular to specifically tailor the number and distribution of sulfate groups and/or anhydro-bridges in carrageenan. The high purity sulfohydrolases of the present invention are especially useful in tailoring properties.
Possibly, the most direct application of this discovery is substitution of enzymatic modification for existing chemical modification processes where comparable (or better) functionality, e.g., gelling properties, viscosity, texture in food, can be achieved with enzymatic modification.
For example, a carrageenan that contains precursor and therefore does not gel, can be induced to gel enzymatically.
As another example, a nu-carrageenan may be converted into an iota-carrageenan to form a product that does not gel under conditions wherein the product has a concentration of about 0.7% (w/v) in an aqueous solution having 0.1 M of potassium at a temperature of 65xc2x0 C. and a pH of 7.
In addition, and by way of non-limiting example only, the functionality of these enzymes may have the following applications: selective control of the induction of 3,6-anhydrogalactose residues; tailoring those properties of carrageenans which are related to the number and distribution of both the sulfate groups and 3,6-anhydrogalactose residues; inducing modification and gelation of iota-precursor in aqueous systems (including cold gelation of cold-soluble carrageenan solutions and pastes, with representative use including food, pharmaceutical, textile, and personal hygiene products, further including special uses such as dairy products, especially powder formulations, toothpaste, and other paste or slurry products); control of the production of iota carrageenan for gelatin replacement (as the enzyme is expected to give an incomplete modification in comparison with the current alkaline process it will allow better control of the gelatin-like properties which depend on such incomplete modification); modification of only iota-precursor in kappa-iota-precursor mixtures, as for example neutral extracts of cold water seaweed specifies, which may give lower gel strength but increased water-binding capacity as well as other new characteristics; and use with other galactan structures having 6-sulfate groups, including agaroids, as well as with homologous nucleic acids and proteins in carrageenophytes and agarophytes.
The precursor-containing carrageenan may be used by injection into or to coat uncooked meat, e.g., chicken, salmon. If such enzymes are included in the mixture, they will make the carrageenan gel, so that when the meat is cooked, juices are prevented from leaking out of the meat.
Further possible uses include the following:
preparation of high molecular weight carrageenan with high viscosity and high gel strength by using mild conditions for extraction of the carrageenan, followed by use of the enzymes for modification of the carrageenan; and
gelation at or near ambient temperature (cold gelation), reducing or eliminating the need for heating and/or cooling steps in processes, such as yogurt production, that require the production of a gel.